Methylmalonic acid (MMA), a sensitive biomarker of functional vitamin B12 deficiency, is commonly determined by gas chromatography-mass spectrometry (GC-MS) or liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods using manual extraction and derivatization of MMA to reduce polarity prior to separation. Methods In the present study we introduce a semi-automated extraction on a strong anion exchanger, HPLC separation on a BEH-amide column to separate serum MMA from its abundant

more »

... , succinic acid, followed by MS/MS detection and quantification. Results The extraction of MMA plus internal standard provides full recovery and the method is linear between 0.03µmol/L and 20.0 µmol/L (r 2 =1.0) with intra-and inter-assay imprecision of 2.2%. Agreement with other laboratories has been demonstrated in external proficiency testing. Compared to both conventional GC-MS and LC-MS/MS methods, the correlation is r 2 > 0.99. Conclusions The use of robotic pipetting, elimination of derivatization and improved separation by the BEH-amide column combined with HILIC chromatographic conditions significantly improve sample throughput compared to conventional methods. Using a single pipetting robot and LC-MS/MS instrument, this method is currently performing 180 analyses per day from10 regional hospitals and several additional distant sites.